Original Paper Czech J. Anim. Sci., 59, 2014 (3): 140–145

Melatonin receptor (MTNR1A and MTNR2B) expression during the breeding season in the yak (Bos grunniens)

S.-D. Huo1, 2, R.-J. Long1

1College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, P.R. China 2College of Life Science and Engineering, Northwest University for Nationalities, Lanzhou, P.R. China

ABSTRACT: plays key roles in a wide range of mammalian body functions, which are mediated by the melatonin-specific cell surface receptor MTNR1A( and MTNR1B). To better understand the role of MTNR in the yak (Bos grunniens), we determined the melatonin receptor mRNA expression level. The analysis showed that the MTNR mRNA expression level was higher in the tissue than in the , , and (P < 0.01) during the breeding season. Immunofluorescence analyses showed that yak MTNR was located in the pinealocyte, synaptic ribbon, and synaptic spherules of the pineal gland and that melatonin interacts via nerve fibres. In the hypothalamus,MTNR was located in the magnocellular neurons and parvicellular neurons. MTNR was localized in acidophilic cells and basophilic cells in the pituitary gland. In the ovary, MTNR was present in the ovarian follicle, , and Leydig cells.

Keywords: domestic yak; immunofluorescence; MLTR; real-time PCR

The domestic yak (Bos grunniens) is a rare bovine receptor 1A (MTNR1A) genes are expressed in the species found at high altitudes in the Qinghai- cumulus-oocytes complex, whereas the MTNR1B Tibetan Plateau and adjacent regions. Yaks have gene is expressed only in the oocytes. Melatonin unique biological and economic characteristics that can enhance the maturation of oocytes in vitro make them resistant to cold. They have evolved a (Berlinguer et al., 2009; Kang et al., 2009; El-Raey et unique body structure and physiological adapta- al., 2011). There was a positive correlation between tion mechanisms. The reproductive mechanism MTNR1A allele polymorphisms and reproduction may be the most important requirement for yak in the Sarda sheep breed (Carcangiu et al., 2009, survival in this situation. The yak breeding season 2011). Concluding data revealed the positive ef- runs from July to January in China. This period fect of melatonin treatment on the time of the is perfectly synchronized with the long nights first conception in ewe lambs and highlighted that and short days between the summer solstice and +/+ genotype is able to influence reproductive re- winter solstice. sponse to melatonin treatment. Melatonin-treated The pineal gland is the only endocrine organ animals of +/+ genotype showed a higher number that can transform light into endocrine informa- of pregnancies and lambed earlier compared to tion (Binkley, 1993). Melatonin is a polypeptide untreated animals of the same genotype in MTNR1 , which is synthesized primarily in the (Mura et al., 2010). Melatonin cycle pineal gland. The day length is the main factor varied under different lighting conditions (Ogino to initiate the yak breeding season each year via et al., 2013). There is no doubt that melatonin melatonin. Melatonin has an important role in participates in reproduction but the process is the maturation of oocytes where the melatonin not clear. We hypothesized that melatonin may

Supported by the National Natural Science Foundation of China (Project No. 31260589).

140 Czech J. Anim. Sci., 59, 2014 (3): 140–145 Original Paper regulate yak reproduction via the hypothalamus- 5 min, 95°C for 10 s, and 60°C for 30 s. The cycle pituitary gland- axis. threshold (Ct) was returned to the baseline dur- In this study, we report the expression of MTNR ing each reaction. Based on the melting curve, we in the pineal gland, hypothalamus, pituitary gland, determined whether a specific product or a primer and ovary of the yak. We also examined differences dimer was present in the PCR during each reaction. in the tissue-specific mRNA expression level of the The standard curves were generated from a stand- MTNR gene in the yak during the breeding season. ard sample, i.e. a 10-fold serial dilution (from 101 to 106). The standard curve parameter calculations MATERIAL AND METHODS were used to determine the correlation coefficient 2 value: R MTNR1A = 0.997, slope = –3.387, intercept = 2 Sample collection and preservation. Tissue 28.833; R MTNR2B = 0.997, slope = –3.417, intercept = 2 samples from the pineal gland, hypothalamus, pi- 29.462; R β-actin = 0.999, slope = –3.389, intercept = tuitary gland, and ovary were obtained at the time 16.654. Each sample was tested in triplicate. of slaughter and stored immediately in RNAlater Immunohistochemical analysis of MTNR pro- (Omega Bio-Tek, Inc., Norcross, USA) and para- tein expression. An immunohistochemical assay formaldehyde until further use. Samples were was developed to study MTNR protein expres- collected from six yaks (proestrus) on October 4, sion in yak tissues (pineal gland, hypothalamus, 2012, which was during the breeding season. pituitary gland, and ovary) during the breeding RNA isolation and reverse transcription-poly- season. The tissues were embedded in paraffin merase chain reaction (RT-PCR). Total tissue and dehydrated. MTNR protein expression was RNA was extracted using RNAiso Plus (TaKaRa analyzed using an SP immunohistochemistry kit Biotechnology Co., Dalian, P.R. China) and the (Maixin, Fuzhou, P.R. China), according to the quality was checked with UV-800 ultraviolet spec- manufacturer’s instructions. trophotometer (Shimadzu Corp., Kyoto, Japan) in Statistical analysis. The data were expressed A260/280 nm. Reverse transcription was performed as the mean ± SD. The SPSS software package using PrimeScript RT Master Mix Perfect Real (Version 15.0, 2007) was used to analyze the data. Time (TaKaRa) in a reaction mixture volume of ANOVA was used to test for significant differences 10 μl at 37°C for 15 min and 85°C for 5 s. between different groups. Real-Time PCR of MTNR mRNA expression. To compare MTNR mRNA expression in different yak RESULTS tissues (pineal gland, hypothalamus, pituitary gland, and ovary) during the breeding season, a real-time Tissue-specific expression of the yak mela- PCR assay was developed based on SXBR Premix tonin receptor during the breeding season. The Ex TaqTM II (Perfect Real Time) (TaKaRa). The MTNR1A and MTNR1B mRNA expression levels β-actin was used as a housekeeping gene to cor- were higher in pineal gland tissue compared with rect potential variations in the RNA loading. The the hypothalamus, pituitary gland, and ovary (P < primers were designed using Primer Premier 6.0 0.01) during the yak breeding season (Figure 1). based on the mRNA sequence of MTNR1B. Tau- rus (NCBI Accession numbers XM614283.3 and 1.2 XM001254949.3). The primers used for MTNR1A 1.0 MT1 MT2 were 5'-GGCACTCGTCATCATTCC-3' (forward) 0.8 and 5'-CGTCCACTCCAGTCTTCT-3' (reverse) 0.6 while those used for MTNR1B were 5'-TCGTC- 0.4 TATGGGCTCCTGAA-3' (forward) and 5'-GCT- 0.2 GCCCTTGGAAGAGTT-3' (reverse). The primers 0.0 used for the β-actin plasmid were 5'-CACAGC- Pineal gland Hypothalamus Pituitary gland Ovary CGAGCGGGAAAT-3' (forward) and 5'-CCGT- GTTGGCGTAGAGGT-3' (reverse) (Zi et al., 2012). Figure 1. MTNR1A and MTNR2B mRNA expression in Real-time PCR conditions: 12.5 μl SXBR Premix pineal gland, hypothalamus, pituitary gland, and ovary Ex TaqTM II (2×), 2.0 μl of template cDNA, 1.0 μl tissue of the yak during breeding season (n = 6) as deter- of each primer (10 μmol/l), 8.5 μl deionized H2O. mined by real-time PCR. Values are normalized with the The PCR parameters were 50°C for 2 min, 95°C for values for β-actin. Each sample was analyzed in triplicate 141 Original Paper Czech J. Anim. Sci., 59, 2014 (3): 140–145

Figure 2. Melatonin receptor protein expressions in pineal Figure 3. Melatonin receptor protein expression in hypo- gland tissue of the yak during breeding season (n = 6) as issue of the yak during breeding season (n = 6) as determined by immunofluorescence. The pineal gland, determined by immunofluorescence. Melatonin receptor pinealocyte (PC), synaptic ribbon (SR), and synaptic was obviously located in magnocellular neurons (MN) spherules (SS) were the receptors of melatonin and parvicellular neurons (PN)

There were no significant differences between the Melatonin receptor protein expression in the hypothalamus, pituitary gland, and ovary tissue. pituitary gland. Low MTNR expression was de- Melatonin receptor protein expression in the tected in the pituitary gland tissue α-cells and pineal gland. The pineal gland is the main tissue β-cells during the yak breeding season (Figure 4). where melatonin was produced. The pinealocyte, Melatonin receptor protein expression in the synaptic ribbon (SR), and synaptic spherules (SS) ovary. In the ovary, MTNR expression was high expressed melatonin in the pineal gland (Figure 2). in the ovarian follicle (Figure 5A) but lower in the This suggests that the pinealocytes exchanged in- corpus luteum (Figure 5B) than in Leydig cells. formation via the SR and SS. Thus, the pinealocytes MTNR expression was detected in granular leu- produced melatonin and they also received it. kocytes and oocytes. This suggests that melatonin Melatonin receptor protein expression in the was involved with the growth and maturation of hypothalamus. High MTNR expression was de- ovarian follicles and it also participated in the tected in the magnocellular neurons and parvicel- growth of oocytes. Melatonin also stimulated lular neurons of the hypothalamus tissue during hormone production by Leydig cells. MTNR was the breeding season (Figure 3). expressed by the granulosa cells in the follicle during each stage.

DISCUSSION

This study investigated MTNR gene expression in the yak (B. grunniens). We examined tissue- specific differences in the MTNR mRNA expression levels in the yak during the breeding season. We also studied various characteristics of the tissue- specific MTNR protein expression in the yak. To the best of our knowledge, this is the first study to report the tissue-specific MTNR mRNA and protein expression levels in the yak. The yak is a seasonal breeder with a low repro- Figure 4. Melatonin receptor protein expression in α-cells ductive rate and milk yield compared with dairy (α) and β-cells (β) of pituitary gland tissue during breeding cattle breeds (Zi, 2003; Sarkar et al., 2005; Reiter season (n = 6). The colloid lost in the folliculi (F) et al., 2009). Previous studies have shown that 142 Czech J. Anim. Sci., 59, 2014 (3): 140–145 Original Paper

and ovary during the breeding season in yak, in- dicating that melatonin may have a critical role in hormonal production during the yak breeding season. Melatonin has direct effects on tissues such as the hypothalamus, pituitary gland, and ovary. We hypothesized that higher melatonin production would stimulate the tissue to produce hormone. However, the melatonin level would have to exceed a threshold amount before would be produced in tissues. In the first 30 years since the discovery of indole, much of the research related to melatonin and the pineal gland was related to its ability to modulate the reproductive physiology of photoperiod-de- pendent seasonally breeding mammals (Reiter et al., 1966; Reiter, 1973; Stetson et al., 1975; Turek et al., 1976). Thus, we determined the localization of melatonin and the cells with melatonin recep- tors. Positive staining for MTNR was detected in the pineal gland, pinealocyte, SR, and SS (Fig- ure 2). The pineal gland immunohistochemistry detected a circular pattern in the pineal gland cells. We suggest that the pinealocytes produced melatonin and that melatonin could activate other pinealocytes and the SR and SS in other regions of the pineal gland. This stimulation may have produced more melatonin in pinealocytes. The hypothalamus mainly produces hormones related to reproduction, such as GnRH. GnRH is produced by the hypothalamus and it helps to synchronize ovulation with sexual behaviour (Marshall et al., 1980). Ovulation occurred as early as 15 days postpartum with 1.0 mg GnRH (Pinheiro et al., 2013). Melatonin receptors were observed in mag- nocellular neurons and parvicellular neurons in the hypothalamus, and mRNA expression was lower than that in the pineal gland (P < 0.01). However, this did not show that it was absolutely lower in the hypothalamus. High MTNR expression was Figure 5. Melatonin receptor protein expression in oocyte observed in the magnocellular neurons and par- (O), granulocyte (G), corpus luteum (CL), and Leydig cells vicellular neurons (Figure 3) but it was unclear (L) of pituitary gland tissue during breeding season (n = 6) whether hormonal production was stimulated or inhibited in these cells. It is clear that melatonin melatonin receptor protein expression in antral follicle (A), takes part in the generation of GnRH. We suggest in primordial follicle and primary ovarian follicle (B), and that it may increase one type of hormone and de- in corpus luteum (C) crease another type of hormone in the hypothala- AF = autrum of follicle, PF = primordial follicle, POF = mus. MTNR protein was observed in the α-cells primary ovarian follicle and β-cells of the pituitary gland (Figure 4). LH and FSH are the main hormones produced in the melatonin has a critical role during reproduction pituitary gland for reproduction. GnRH could affect as a neural signal in mammals. MTNR mRNA was LH and FSH via the hypothalamus-pituitary axis expressed in the hypothalamus, pituitary gland, to adjust the progress of reproduction (Emanuele 143 Original Paper Czech J. Anim. Sci., 59, 2014 (3): 140–145 et al., 1987). Melatonin significantly reduced the opmental competence and blastocyst output in a goat plasma levels of LH and 17-β-estradiol, while uri- model. Journal of Pineal Research, 46, 383–391. nary 6-sulfatoximelatonin (STM) was increased Binkley S. (1993): Structures and molecules involved in at the morning estrus (Chuffa et al., 2011). Im- generation and regulation of biological rhythms in ver- munohistochemical analysis of the pituitary gland tebrates and invertebrates. Experientia, 49, 648–653. showed that the sides of the folliculi had variable Carcangiu V., Mura M.C., Vacca G.M., Pazzola M., Dettori levels of MTNR expression, which were distinct in M.L., Luridiana S., Bini P.P. (2009): Polymorphism of the the pituitary gland. We suggest that melatonin and melatonin receptor MT1 gene and its relationship with GnRH adjust the hormones in the pituitary gland. seasonal reproductive activity in the Sarda sheep breed. GnRH operates via the hypothalamus-pituitary Animal Reproduction Science, 116, 65–72. axis to participate in precession and melatonin is Carcangiu V., Luridiana S., Vacca G.M., Daga C., Mura M.C. present. In the ovary, we found that MTNR expres- (2011): A polymorphism at the melatonin receptor 1A sion was higher in the ovarian follicle (Figure 5A, B) (MTNR1A) gene in Sarda ewes affects fertility after AI and lower in the corpus luteum (Figure 5C) than in the spring. Reproduction, Fertility, and Development, in Leydig cells. MTNR expression was observed in 23, 376–380. the granular leukocytes and oocytes. We obtained Chuffa L.G., Seiva F.R., Favaro W.J., Teixeira G.R., Amorim similar results like Tamura et al. 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Corresponding Author Sheng-dong Huo, Lecturer, Lanzhou University, College of Pastoral Agriculture Science and Technology, Tianshui South Road 222, 730 00 Lanzhou, P.R. China Phone: +86 18 909 463 712, fax: +86 09 312 938 199, e-mail: [email protected]

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